The evolution and survival of higher life forms on Earth has relied upon an oxygen-enriched atmosphere to drive efficient energy metabolism. Physiological and behavioral mechanisms have evolved to maintain adequate levels of respiratory oxygen while at the same time limiting toxic reactive oxygen species formation. The ability of all organisms, whether simple or complex, to monitor external and internal oxygen levels is key to survival under normal environmental conditions. In clinical situations where patients may be confronted with pathological conditions such as cardiovascular disease, cancer or respiratory illness, these mechanisms can be disrupted or pushed to their limits. Essential oxygen-sensing mechanisms are diverse and as yet poorly understood. Use of the broad range of experimental tools available in model genetic systems such as C. elegans and Drosophila can contribute significantly to advancing our understanding of mechanisms involved in oxygen-sensing. We have utilized genetic, molecular and physiological approaches to relate a fundamental aerotactic behavior in Drosophila larvae to the activation of a subset of peripheral sensory neurons. Preliminary studies suggest that larvae utilize an oxygen-sensing mechanism to coordinate behaviors necessary for food exit prior to pupariation and ultimate survival. These larval aerotactic preferences require function of a small subset of peripheral sensory neurons specifically expressing the DEG/ENaC ion channel subunit, Pickpocket1 (PPK1). Transgenic hypersensitization of ppk1-expressing neurons causes a dramatic increase in larval sensitivity to increased oxygen levels. Transgenic inactivation of ppk1-expressing neurons using tetanus toxin causes a loss of aversion to increased oxygen levels. We propose a model in which PPK1-expressing neurons may function as sensors of environmental oxygen during larval stages. In arthropods such as Drosophila, oxygen is supplied to essentially every cell and tissue by diffusion of air through a complex tracheal tubule system. During larval foraging stages, when larvae display the highest aversion to increased oxygen levels, the external openings of the larval tracheal system, the posterior spiracles, are the only part of the larva projected above the surface of the food to allow access to the atmosphere. Preliminary results show that a single ppk1-expressing neuron innervates each of the posterior spiracles. The focus of this proposal will be on those posterior spiracle neurons and their potential role in oxygen-sensing and aerotactic behavior. Specific Aim I will utilize Ca2+sensitive G-CaMP imaging techniques to examine direct oxygen-dependent activation of the PPK1-expressing neurons innervating the posterior spiracles. Specific Aim II will seek to correlate the function of the same PPK1-expressing posterior spiracle neurons with larval aerotactic behavior by generating single cell hypersensitized neuronal clones using the MARCM technique. Successful completion of these aims should establish a versatile and productive genetic model for use in future genetic and molecular studies of environmental oxygen sensing mechanisms. PUBLIC HEALTH RELEVANCE: The ability of all organisms, whether simple or complex, to monitor external and internal oxygen levels is key to survival under normal environmental conditions. In clinical situations where patients may be confronted with pathological conditions such as cardiovascular disease, cancer or respiratory illness, these mechanisms can be disrupted or pushed to their limits. Despite the central role of oxygen sensing in the survival of life on this planet, we know relatively little concerning the diverse mechanisms that are utilized to balance oxygen supply with metabolic demand.